The present invention relates to muteins (hereinafter, sometimes to be referred to as mutated molecules) with less epithelial cell growth promoting activity (EGF activity) such as for smooth muscle cells while preserving activity in differentiating betacellulin (BTC) into pancreatic xcex2 cells (BTC activity), as well as a manufacturing method thereof, etc.
Human betacellulin is a protein factor consisting of 80 amino acids excised from a precursor consisting of 178 amino acids. The entire amino acid sequence has been identified (Sasada et al, Biochem. Biophys. Res. Commun., 190:1173 (1993)). Endogenous betacellulin occurs in extremely minute amounts, and it is difficult to obtain the endogenous betacellulin. However, a method for manufacture using a recombinant technique has been disclosed in Japanese Unexamined Patent Application (Kokai) H6-87894, etc. Betacellulin was first discovered as a factor having mouse 3T3 cell growth promoting activity, and has subsequently been found to have vascular smooth muscle cell and retinal pigment epithelial cell growth promoting activity (EGF activity) (Shing et al, Science, 259:1604 (1993)). Betacellulin also acts on pancreatic undifferentiated cells, promoting their differentiation to pancreatic xcex2 cells which produce insulin (BTC activity) (Mashima et al, J. Clinic. Invest., 97:1647 (1996)), and is thus considered to be a potentially useful as prophylactic and therapeutic agent for diabetes (such as insulin-dependent diabetes) as well as pancreatic dysfunction and the like associated with diabetes (Miyagawa et al, Abstracts of the 1997 Japan Diabetes Association Conference, 125).
However, since betacellulin has such vascular smooth muscle cell and retinal pigment epithelial cell growth promoting activity, these activities have proven to be a problem in applications as a therapeutic agent for diabetes.
The potential of such pharmaceuticals could therefore be improved if the growth promoting activity of the vascular smooth muscle cells and the like could be reduced while preserving the action in promoting pancreatic xcex2 cell differentiation, but no such betacellulin muteins are thus far known.
As a result of extensive research on betacellulin muteins, the inventors perfected the present invention upon the discovery and further research of muteins in which betacellulin mutations allowed the smooth vascular muscle growth promoting activity and the like to be reduced while preserving the pancreatic xcex2 cell differentiation promoting activity, with no antigenicity-related problems when administered to the living body. That is, the present invention relates to betacellulin muteins, a method for obtaining said muteins and the like
That is, the present invention relates to:
1. A betacellulin mutein or a salt thereof, wherein the pancreatic xcex2 cell differentiation promoting activity is preserved, and the epithelial cell growth promoting activity is reduced.
2. A betacellulin mutein or salt thereof according to 1 above, wherein the ratio of the pancreatic xcex2 cell differentiation promoting activity to the epithelial cell growth promoting activity is at least twice relative to that of betacellulin.
3. A betacellulin mutein or salt thereof according to 1 above, wherein 1 to 40 amino acid residues from the N terminal of the betacellulin may be deleted, and 1 to 4 of the first through fourth amino acid residues from the C terminal, including the Leu at 3 from the C terminal and the Asp at 4 from the C terminal, may be deleted or substituted with other amino acid residues or other peptide chains.
4. A betacellulin mutein or salt thereof according to 3 above, wherein 1 to 40 amino acid residues from the N terminal have been deleted.
5. A betacellulin mutein or salt thereof according to 3 above, comprising 1) an amino acid sequence represented by SEQ ID NO: 1, 2) an amino acid sequence in which 1 to 40 amino acids from the N terminal in the amino acid sequence represented by SEQ ID NO: 1 have been deleted, 3) an amino acid sequence represented by SEQ ID NO: 2, or 4) an amino acid sequence in which 1 to 40 amino acids from the N terminal in the amino acid sequence represented by SEQ ID NO: 2 have been deleted.
6. A betacellulin or salt thereof according to 3 above, comprising 1) an amino acid sequence represented by SEQ ID NO: 1, 2) an amino acid sequence represented by SEQ ID NO: 2, 3) an amino acid sequence represented by SEQ ID NO: 3, or 4) an amino acid sequence represented by SEQ ID NO: 4.

7. A betacellulin or salt thereof according to 3 above, comprising 1) an amino acid sequence represented by SEQ ID NO: 37, or 2) an amino acid sequence represented by SEQ ID NO: 38.
8. A betacellulin or salt thereof according to 1 above, wherein 1 to 30 amino acid residues from the N terminal of the betacellulin may be deleted, and 1 to 5 amino acid residues may be inserted between the 22nd and 23rd amino acid residues from the C terminal.
9. A betacellulin or salt thereof according to 8 above, wherein 1 to 30 amino acid residues from the N terminal of the betacellulin have been deleted.
10. A betacellulin or salt thereof according to 8 above, comprising the amino acid sequence represented by SEQ ID NO: 44.
11. A betacellulin or salt thereof according to 8 above, comprising the amino acid sequence represented by SEQ ID NO: 45.
12. A method for manufacturing a betacellulin or salt thereof according to 1 above, characterized by culturing the transformants which have been transformed with recombinant vectors containing DNA encoding the betacellulin according to 1 above to produce said betacellulin mutein.
13. A pharmaceutical composition comprising a betacellulin or salt thereof according to 1 above.
14. A pharmaceutical composition according to 9 above, wherein the composition is a prophylactic or therapeutic drug for diabetes.
15. A prophylactic or therapeutic method for diabetes, characterized in that a betacellulin or salt thereof according to 1 above is administered to mammals.
16. The use of a betacellulin or salt thereof according to 1 above to manufacture a prophylactic or therapeutic agent for diabetes.